Blood pressure regulation relies on the ability of membrane proteins to transduce variations in physical stimuli (e.g., hemodynamic forces) into electrical signals. The transient receptor potential vanilloid 4 (TRPV4) is a pu- tative mechanosensitive Ca2+ channel expressed in endothelial and smooth muscle cells and in perivascular sensory neurons. Although TRPV4 has been implicated in endothelium- and perivascular nerve-dependent vasorelaxation, its precise gating mechanism remains elusive. Three mechanisms have been proposed to activate TRPV4 after mechanical stimulation: 1) downstream of the phospholipase A2 (PLA2)-dependent formation of omega (w)-6 arachidonic acid (AA) and its metabolites, epoxyeicosatrienoic acids (EETs); 2) downstream of purinergic P2Y2 receptor activation, mediated by adenosine triphosphate release; and 3) direct activation by membrane stretch. Our long-term goal is to delineate the mechanisms by which ion channels decode exogenous and endogenous stimuli to regulate cellular function. In this proposal, the overall objective is to establish the molecular basis underlying TRPV4 activation. The central hypothesis is that TRPV4 activation is regulated by the mechanical properties of the membrane via lipid remodeling. The rationale for the proposed research plan is that once the precise mechanism of TRPV4 activation has been elucidated, it will be possible to define strategies that target TRPV4 to control systemic blood pressure. The hypothesis will be tested by pursuing three Specific Aims: 1) Determine the effect of w-6 and w-3 fatty acids on TRPV4 activity in C. elegans; 2) Test the hypothesis that w-3 fatty acid derivatives enhance TRPV4 activity in vascular cells; and 3) Determine how changes in the mechanical properties of the membrane regulates TRPV4 gating. We will leverage genetic, behavioral, functional, biochemical, and biophysical approaches to uncover the contribution of fatty acids and their metabolites to TRPV4 function. The research plan is innovative because it will determine the individual contribution of w-3 and w-6 fatty acids and their eicosanoid derivatives to TRPV4 gating. The proposed research is significant because it is expected to have broad translational importance in targeting TRPV4 to regulate vascular and neuronal function.